The present invention relates to a method for producing a protein in a cell-free system, and more particularly, to a method for producing a wide range of proteins used in industrial fields related to pharmaceuticals, food, agricultural chemicals, environmental products and commodity products, in a cell-free system.
Generally speaking, there are two methods for producing protein: via a chemical process and via a biological process. In the case of producing a peptide with few amino acids, a chemical process is mainly used. Particularly, for peptides having ten or fewer amino acids, the chemical process is known to be more economical than the biological process.
Conventionally, protein production by the biological process is accomplished in an in vivo system using a genetically manipulated biological cell. Here, according to the excretion characteristics of the protein, that is, whether it is an intracellular protein or an extracellular protein, one or the other of two methods is used. In one method, an intracellular protein is produced by a method comprising the steps of culturing a cell in a proper medium to accumulate a desired protein in the cell, harvesting the cell at the appropriate growth stage of the cell, rupturing the cell and finally isolating and purifying the desired protein. In another method, an extracellular protein is produced by culturing a cell in a proper broth medium, separating cultural broth without the cell, and isolating and purifying a desired protein.
Such a biological process based on biological cells in a living state has several problems in view of protein over-production by the living biological cells and isolation and purification of a desired protein.
Generally, protein is degraded or modified by several enzymes synthesized with the growth of the cell. After synthesis, protein is frequently modified into undesired forms due to post-translational processing such as deamination or oxidation. It is very difficult to incorporate modified or unnatural amino acids into protein. Also, cytotoxic proteins inhibit the growth of the cell.
The over-production of protein beyond a predetermined concentration is also difficult to obtain because the expression of a gene coding a desired protein may be regulated by the concentration thereof. Even though an artificially mutated cell capable of over-producing the desired protein is used, there is a limitation in producing protein due to the inherent characteristics of the biological cell itself. That is, the concentration of protein accumulated in the cell or excreted into a broth generally affects the viability of the cell. Accordingly, it is very difficult to harmonize the conditions of protein over-production and cell growth, so that over-production of the desired protein is very difficult to obtain.
In an isolation and purification process, many kinds of protein are insoluble or unstable, and are either degraded by intracellular proteases or aggregate in inclusion bodies, so that the loss rate of the desired protein is generally high during protein purification, and particularly, the isolation of membraneous protein is highly complex and difficult. Also, in the case of protein used in protein products such as pharmaceuticals and food, great caution should be taken in order to prevent contamination by infecting agents or endotoxins. Therefore, the efficiency of isolation and purification of the desired protein is poor and the specific production rate and the overall productivity of the protein are both low, so that the price of the protein is considerably high.
In order to solve the above problems appearing in the in vivo system, a method for producing protein in a cell-free system, that is, an in vitro system based on a cell-free extract. However, according to a method using a conventional batch system, the required amount of mRNA is high, the length of an operation cycle is less than one hour, the basic activity of the cell-free extract is very low, and disadvantages due to enzymes degrading nucleic acids or protein are still serious. Further, it is difficult to prevent inhibition caused by energy sources, products or byproducts. Thus, protein productivity remains very low.
In order to solve the above problems caused by the batch process, Alexander S. Spirin et al. have suggested a method of cell-free translation for producing protein using a continuous system (see "A Continuous Cell-free Translation System Capable of Producing Polypeptides in High Yield," Science, Vol. 242, 1988, pp 1162-1164). Using this system, protein synthesis is maintained at a constant rate for more than twenty hours, but the productivity was only about 5 pmol/pmol mRNA. Subsequently, a coupled transcription-translation process with a continuous system was suggested by Vladimir I. Baranov et al. (see "Gene expression in a cell-free system on the preparative scale," Gene, Vol. 84, 1989, pp 463-466). This system worked at a constant rate for tens of hours with protein productivity of 4 .mu.g/ml per hour, resulting in production of preparative amounts of protein. Protein produced in this system was identified by autoradiography after electrophoresis. Such research as above has concentrated on extending the period of protein synthesis but not on increasing the rate of protein synthesis.
However, it is very important to shorten the period to yield products, particularly in the cell-free protein synthesis, considering unstable substrates used in the system, such as nucleotide triphosphates and mRNA. In the case of using a membrane, an efficient mixing is inhibited according to the density increase of the reaction mixture with operation time. Further, flow is perpendicular toward the membrane so that a "plugging" phenomenon is frequently generated and thus operation does not continue functioning properly beyond 100 hours.
Further, Hideo Nakano et al. have suggested a method for increasing the rate of protein synthesis in the cell-free system using a cell-free extract concentrated by an ultrafiltration membrane (see "An Increased Rate of Cell-free Protein Synthesis by Condensing Wheat-germ Extract with Ultrafiltration Membranes," Bioscience, Biotechnology, Biochemistry, Vol. 58(4), 1994, pp 631-634). According to this system, the protein productivity was about 30 .mu.g/ml per hour, which is five-fold that obtained by the above continuous-flow cell-free (CFCF) system, but the final amount of synthesized protein was one third of that obtained by the CFCF system and operation time was only six hours.
As shown from the above, both protein productivity and production amount are still low, which is an obstacle in implementing the industrialization of cell-free protein synthesis. Therefore, improvements are greatly required in terms of the total productivity of the protein by increasing the specific production rate and the length of system operation, and in terms of a cost-reduction by regenerating an expensive energy source in order to recycle the regenerated energy source.